An immunoassay method utilizing reactivation of an apoenzyme is known as one of homogeneous competitive enzyme immunoassay (EIA) methods which are useful as methods for detecting low-molecular compounds such as hormones and peptides with high sensitivity. In the immunoassay method, glucose oxidase requiring flavin adenine dinucleotide (FAD) as a coenzyme is conventionally used as an apoenzyme (Patent Document 1, Non-Patent Documents 1 to 3).
However, as described in Non-patent Document 3, the detection limit of trinitrotoluene in the method using glucose oxidase is 5 μg/L (5 ng/mL), that is, the method has a problem that only limited targets can be assayed therewith due to its insufficient sensitivity.
Furthermore, there are also problems that a blank can be varied because the apoenzyme can react with not only glucose added as a substrate but also endogenous substrates or coenzymes such as glucose or free FAD derived from a test sample, resulting in reaction between a detection reagent and hydrogen peroxide produced nonspecifically, and the like.
[Patent Document 1] JP-A-55-2997
[Non-Patent Document 1] Anal. Chem., 1981, 658-665
[Non-Patent Document 2] Meth. Enzymol., 1983, 413
[Non-Patent Document 3] Fresenius J. Anal. Chem., 1998, Vol. 361, 174-178